Cross-link defects in collagen are characteristics of a number of fatal, incapacitating, or severely disfiguring diseases such as Marfans syndrome, Ehlers-Danlos syndrome and Menkes syndrome. Collagen cross-linking is among the sequelae of diabetes mellitus and may be important in accelerated atherosclerosis and diabetic cataracts. The nature of collagen cross-links and their maturation is poorly understood in part because of the inadequacy of methods to study them directly in native collagen. The work proposed here addresses the observation and characterization of rat tail collagen cross-links as a function of the maturation process. Methodological development will be followed in the long term to applications to studies of the effects of disease states on native collagen and the effects of drugs such as lysyl oxidase inhibitors on collagen cross-linking. Collagen cross-links will be labeled with stable-isotope derivatives of lysine, proline and histidine; and the cross-links will be observed and characterized by solids nuclear magnetic resonance (NMR) methods previously applied in our laboratories to bacterial cell wall cross-linking. Cross-polarization NMR with magic angle spinning (CPMAS NMR) will be used to detect and quantify the 13C- and 15N-labeled cross-links; and double cross polarization (DCPMAS) techniques will be used to observe and quantify direct 13C -15N bonds. Using these techniques the allysine pathway for cross-linking in rat tail collagen will be tested.